Aversive stimuli bias corticothalamic responses to motivationally significant cues.

Making predictions about future rewards or punishments is fundamental to adaptive behavior. These processes are influenced by prior experience. For example, prior exposure to aversive stimuli or stressors changes behavioral responses to negative- and positive-value predictive cues. Here, we demonstrate a role for medial prefrontal cortex (mPFC) neurons projecting to the paraventricular nucleus of the thalamus (PVT; mPFC→PVT) in this process. We found that a history of aversive stimuli negatively biased behavioral responses to motivationally relevant cues in mice and that this negative bias was associated with hyperactivity in mPFC→PVT neurons during exposure to those cues. Furthermore, artificially mimicking this hyperactive response with selective optogenetic excitation of the same pathway recapitulated the negative behavioral bias induced by aversive stimuli, whereas optogenetic inactivation of mPFC→PVT neurons prevented the development of the negative bias. Together, our results highlight how information flow within the mPFC→PVT circuit is critical for making predictions about motivationally-relevant outcomes as a function of prior experience.

Low semen quality and adverse histological changes in testes of adult male mice treated with bee venom (Apis mellifera).

Background: Male infertility has been on the rise since the past seven decades. Recently, in Libya, bee venom therapy (BVT) has become a popular method among alternative healthcare practitioners for treating male infertility. However, a literature search did not find any published studies that investigated the use of BVT for infertility treatment. Aim: To investigate the effect of bee venom on the male reproductive status through measurements of semen quality parameters and testicular histological changes in adult male mice. Methods: A total of 48 male mice were randomly divided into three experimental groups (which were subdivided into two subgroups with eight mice each) as follows: control, bee venom sting (BVS), and bee venom injection (BVI). The normal control subgroup mice were not subjected to any treatment, while the vehicle control subgroup mice were injected (i.p.) with 200 µl of 0.9% saline solution. In the BVS-treated subgroups, each mouse was stung by one live bee for five times (BVS-5) or seven times (BVS-7) every third day for 2 or 3 weeks. While each mouse in the BVI-treated subgroups received 23 µg/kg in a dose volume of 200 µl BVIs (i.p.) for five times (BVI-5) or seven times (BVI-7) every third day for 15 or 21 days. Results: The findings of this study showed that repeated bee venom treatment by sting or injection to adult male mice resulted in a significant decline in testosterone levels, sperm count, sperm motility, and a very significant increase in the percentage of abnormal sperm morphology; also, there were harmful testicular histological changes in the structural organization of seminiferous tubules and degenerative changes in the germinal epithelium compared to control group. Conclusion: The results of this study provide evidence for the low semen quality and adverse testicular histological changes in male mice treated with bee venom. Hence, there is a desperate need for educating alternative healthcare practitioners and infertile couples about the harmful effects of BVT on reproductive status.

Selenium supplementation inhibits IGF-1 signaling and confers methionine restriction-like healthspan benefits to mice.

Methionine restriction (MR) dramatically extends the healthspan of several organisms. Methionine-restricted rodents have less age-related pathology and increased longevity as compared with controls, and recent studies suggest that humans might benefit similarly. Mechanistically, it is likely that the decreased IGF-1 signaling that results from MR underlies the benefits of this regimen. Thus, we hypothesized that interventions that decrease IGF-1 signaling would also produce MR-like healthspan benefits. Selenium supplementation inhibits IGF-1 signaling in rats and has been studied for its putative healthspan benefits. Indeed, we show that feeding mice a diet supplemented with sodium selenite results in an MR-like phenotype, marked by protection against diet-induced obesity, as well as altered plasma levels of IGF-1, FGF-21, adiponectin, and leptin. Selenomethionine supplementation results in a similar, albeit less robust response, and also extends budding yeast lifespan. Our results indicate that selenium supplementation is sufficient to produce MR-like healthspan benefits for yeast and mammals.

Role of zinc in female reproduction.

Zinc is a critical component in a number of conserved processes that regulate female germ cell growth, fertility, and pregnancy. During follicle development, a sufficient intracellular concentration of zinc in the oocyte maintains meiotic arrest at prophase I until the germ cell is ready to undergo maturation. An adequate supply of zinc is necessary for the oocyte to form a fertilization-competent egg as dietary zinc deficiency or chelation of zinc disrupts maturation and reduces the oocyte quality. Following sperm fusion to the egg to initiate the acrosomal reaction, a quick release of zinc, known as the zinc spark, induces egg activation in addition to facilitating zona pellucida hardening and reducing sperm motility to prevent polyspermy. Symmetric division, proliferation, and differentiation of the preimplantation embryo rely on zinc availability, both during the oocyte development and post-fertilization. Further, the fetal contribution to the placenta, fetal limb growth, and neural tube development are hindered in females challenged with zinc deficiency during pregnancy. In this review, we discuss the role of zinc in germ cell development, fertilization, and pregnancy with a focus on recent studies in mammalian females. We further detail the fundamental zinc-mediated reproductive processes that have only been explored in non-mammalian species and speculate on the role of zinc in similar mechanisms of female mammals. The evidence collected over the last decade highlights the necessity of zinc for normal fertility and healthy pregnancy outcomes, which suggests zinc supplementation should be considered for reproductive age women at risk of zinc deficiency.

Effect size, sample size and power of forced swim test assays in mice: Guidelines for investigators to optimize reproducibility.

A recent flood of publications has documented serious problems in scientific reproducibility, power, and reporting of biomedical articles, yet scientists persist in their usual practices. Why? We examined a popular and important preclinical assay, the Forced Swim Test (FST) in mice used to test putative antidepressants. Whether the mice were assayed in a naïve state vs. in a model of depression or stress, and whether the mice were given test agents vs. known antidepressants regarded as positive controls, the mean effect sizes seen in the experiments were indeed extremely large (1.5-2.5 in Cohen's d units); most of the experiments utilized 7-10 animals per group which did have adequate power to reliably detect effects of this magnitude. We propose that this may at least partially explain why investigators using the FST do not perceive intuitively that their experimental designs fall short-even though proper prospective design would require ~21-26 animals per group to detect, at a minimum, large effects (0.8 in Cohen's d units) when the true effect of a test agent is unknown. Our data provide explicit parameters and guidance for investigators seeking to carry out prospective power estimation for the FST. More generally, altering the real-life behavior of scientists in planning their experiments may require developing educational tools that allow them to actively visualize the inter-relationships among effect size, sample size, statistical power, and replicability in a direct and intuitive manner.

A Novel Micronutrient Blend Mimics Calorie Restriction Transcriptomics in Multiple Tissues of Mice and Increases Lifespan and Mobility in C. elegans.

BACKGROUND: We previously described a novel micronutrient blend that behaves like a putative calorie restriction mimetic. The aim of this paper was to analyze the beneficial effects of our micronutrient blend in mice and C. elegans, and compare them with calorie restriction. METHODS: Whole transcriptomic analysis was performed in the brain cortex, skeletal muscle and heart in three groups of mice: old controls (30 months), old + calorie restriction and old + novel micronutrient blend. Longevity and vitality were tested in C. elegans. RESULTS: The micronutrient blend elicited transcriptomic changes in a manner similar to those in the calorie-restricted group and different from those in the control group. Subgroup analysis revealed that nuclear hormone receptor, proteasome complex and angiotensinogen genes, all of which are known to be directly related to aging, were the most affected. Furthermore, a functional analysis in C. elegans was used. We found that feeding C. elegans the micronutrient blend increased longevity as well as vitality. CONCLUSIONS: We describe a micronutrient supplement that causes similar changes (transcriptomic and promoting longevity and vitality) as a calorie restriction in mice and C. elegans, respectively, but further studies are required to confirm these effects in humans.

Individual variability in female and male mice in a test-retest protocol of the forced swim test.

BACKGROUND: The challenges to embody the complexity of symptoms and biological mechanism of affective disorders question the value of animal models as well as their reproducibility and validity. Validity is further hindered by large individual variability in many models. Whereas individual variability presents a challenge, it can also be used to study susceptibility and resistance. One of the frequently used models for screening antidepressants and interventions related to depression is the forced swim test (FST). The FST is typically performed only once. METHODS: The current study was designed with a number of objectives: (1) Examine the group effects of repeated FST (2) Examine the interaction between sex and repeated FST and (3) examine the consistency of individual variability across test and retest in the FST. We exposed ICR female and male mice to the FST 3 or 5 times with two days between exposures. Immobility time was analyzed across exposures at the group and the individual levels using repeated measures ANOVA as well as Pearson's correlations. RESULTS: As expected, repeated exposure to the FST resulted in increased immobility across exposures with no consistent effect of sex. At the level of individual mice, immobility time showed correlation across exposures. DISCUSSION: The current study demonstrates the effects of repeating the FST in both sexes with attention to individual variability. The results suggest that the FST can be used more than once and that mice show a consistent individual pattern of responding in the test.

Effects of Repeated Intraperitoneal Injection of Pharmaceutical-grade and Nonpharmaceutical-grade Corn Oil in Female C57BL/6J Mice.

Due to potential adverse effects on animal wellbeing, the use of nonpharmaceutical-grade substances in animal research must be scientifically justified in cases where a pharmaceutical-grade version of the substance exists. This requirement applies to all substances, including vehicles used to solubilize experimental drugs. To date, no studies have evaluated the direct effect of the pharmaceutical classification of a compound on animal wellbeing. In this study, we evaluated intraperitoneal administration of pharmaceutical-grade corn oil, nonpharmaceutical-grade corn oil, and saline in female C57BL/6J mice. Compounds were administered every 48 h for a total of 4 injections. Mice were evaluated clinically by using body weight, body condition score, visual assessment score, CBC, and serum chemistries. Animals were euthanized at 24 h and 14 d after the final injection. Inflammation of the peritoneal wall and mesenteric fat was assessed microscopically by using a semiquantitative scoring system. Saline-dosed groups had lower pathology scores at both time points. At day 21, pharmaceutical-grade corn oil had a significantly higher pathology score compared with nonpharmaceutical-grade corn oil. No other significant differences between the corn oil groups were observed. The use of nonpharmaceutical grade corn oil did not result in adverse clinical consequences and is presumed safe to use for intraperitoneal injection in mice. Differences in inflammation between the 2 groups suggest that the use of either pharmaceutical-grade or nonpharmaceutical-grade corn oil should be consistent within a study.

A method for reducing environmental pollution by using essential oils in rodent pest management program.

Strong-smelling plant extracts, such as essential oils, have a variety of feeding effects on mammals. Considering current concerns over long-term health issues and environmental effects of chemicals, plant-based products with repellent or antifungal activities may represent good solutions for improvement of rodent pest control programs. The present study was therefore focused on examining the effects of bergamot, lavender, and thyme essential oils as additional bait components on daily intakes of cereal-based baits by wild house mice. Lavender essential oil, containing linalool and linalyl acetate as main components, and thyme essential oil with a prevailing thymol component had no effects on house mice diet. Bergamot essential oil, whose main components were linalool, limonene, and linalyl acetate, showed a repellent effect on house mouse diet.

Locomotor effects of a low-frequency fire alarm on C57BL/6 male mice: a preliminary study.

Maintaining appropriate acoustic conditions for animal welfare and data collection are crucial in biomedical research facilities. Negative impacts of disruptive sound are known and can include auditory damage, immune function changes, and behavioral alterations. One type of disruptive sound occurring in research facilities is that of fire alarms. To ameliorate this problem, many facilities have incorporated the use of low-frequency fire alarms that emit tones outside the rodent audible range. The impact of these devices has been assumed to be negligible. However, this has yet to be evaluated with controlled behavioral experiments. Thus, our objective was to investigate the impact of low-frequency fire alarm exposure on locomotor behavior in the open field, a test sensitive to acoustic stimuli disruption. Male mice were randomized to three alarm exposure groups (No-Alarm; Alarm-During; and Alarm-After) and placed in individual photobeam-activated locomotor chambers. The Alarm-During group displayed significantly reduced horizontal locomotion, with a trend towards reduced vertical locomotion. These data suggest that a low-frequency brief alarm tone can temporarily disrupt movement, a valuable insight should an alarm be deployed. Further, findings support close collaboration between researchers and institutional facility staff to ensure appropriate acoustic conditions are maintained, whenever possible, for research animals.

Evaluation of aggressiveness of female mice using a semi-automated apparatus for measurement of aggressive biting behavior toward an inanimate object.

BACKGROUND: Most laboratory research on aggressive behavior has focused on intraspecific intermale aggression tests. The intraspecific confrontation is not available for the evaluation of female aggressiveness, since androgens are critical for maintenance of this behavior, whereas aggressive biting behavior toward inanimate objects (ABI) occurs in both males and females. NEW METHOD: We propose an experimental method for evaluating female aggressiveness. We improved the previously developed semi-automated apparatus (Aggression Response Meter, ARM) to apply it to measurement of female ABI, and measured changes of ABI in stressed mice and drug actions on ABI. RESULTS: ABI assessment was performed daily in sexually mature female mice using ARM. The intensity and number of ABI in one session did not significantly change during an estrous cycle, suggesting that ABI is not influenced by the dynamics of sex hormones. Additional female mice were socially isolated for 7 weeks and then re-socialized for 2 weeks, and ABI was monitored weekly. ABI significantly increased during the isolation period, and then significantly decreased during re-socialization; both were time-dependent. In prolonged-isolated aggressive mice, a serotonin 1A receptor agonist, buspirone, significantly decreased ABI. COMPARISON WITH EXISTING METHOD: There are no experimental methods or apparatus available for evaluating female aggressiveness using one individual repeatedly. We could measure ABI semi-quantitatively using the ARM. CONCLUSIONS: ABI is a useful behavioral paradigm in the evaluation of aggressiveness in female mice, regardless of the estrous cycle, and can also be used for evaluating the actions of drugs on aggressiveness.

High-Throughput Humanized Mouse Models for Evaluation of HIV-1 Therapeutics and Pathogenesis.

Mice cannot be used as a model to evaluate HIV-1 therapeutics because they do not become infected by HIV-1 due to structural differences between several human and mouse proteins required for HIV-1 replication. This has limited their use for in vivo assessment of anti-HIV-1 therapeutics and the mechanism by which cofactors, such as illicit drug use accelerate HIV-1 replication and disease course in substance abusers. Here, we describe the development and application of two in vivo humanized mouse models that are highly sensitive and useful models for the in vivo evaluation of candidate anti-HIV therapeutics. The first model, hu-spl-PBMC-NSG mice, uses NOD-SCID IL2rγ(-/-) (NSG) mice intrasplenically injected with human peripheral blood mononuclear cells (PBMC) which develop productive splenic HIV-1 infection after intrasplenic inoculation with a replication-competent HIV-1 expressing Renilla reniformis luciferase (HIV-LucR) and enables investigators to use bioluminescence to visualize and quantitate the temporal effects of therapeutics on HIV-1 infection. The second model, hCD4/R5/cT1 mice, consists of transgenic mice carrying human CD4, CCR5 and cyclin T1 genes, which enables murine CD4-expressing cells to support HIV-1 entry, Tat-mediated LTR transcription and consequently develop productive infection. The hCD4/R5/cT1 mice develop disseminated infection of tissues including the spleen, small intestine, lymph nodes and lungs after intravenous injection with HIV-1-LucR. Because these mice can be infected with HIV-LucR expressing transmitted/founder and clade A/E and C Envs, these mouse models can also be used to evaluate the in vivo efficacy of broadly neutralizing antibodies and antibodies induced by candidate HIV-1 vaccines. Furthermore, because hCD4/R5/cT1 mice can be infected by vaginal inoculation with replication-competent HIV-1 expressing NanoLuc (HIV-nLucR)-, this mouse model can be used to evaluate the mechanisms by which substance abuse and other factors enhance mucosal transmission of HIV-1.

Facial whisker pattern is not sufficient to instruct a whisker-related topographic map in the mouse somatosensory brainstem.

Facial somatosensory input is relayed by trigeminal ganglion (TG) neurons and serially wired to brainstem, thalamus and cortex. Spatially ordered sets of target neurons generate central topographic maps reproducing the spatial arrangement of peripheral facial receptors. Facial pattern provides a necessary template for map formation, but may be insufficient to impose a brain somatotopic pattern. In mice, lower jaw sensory information is relayed by the trigeminal nerve mandibular branch, whose axons target the brainstem dorsal principal sensory trigeminal nucleus (dPrV). Input from mystacial whiskers is relayed by the maxillary branch and forms a topographic representation of rows and whiskers in the ventral PrV (vPrV). To investigate peripheral organisation in imposing a brain topographic pattern, we analysed Edn1(-/-) mice, which present ectopic whisker rows on the lower jaw. We found that these whiskers were innervated by mandibular TG neurons which initially targeted dPrV. Unlike maxillary TG neurons, the ectopic whisker-innervating mandibular neuron cell bodies and pre-target central axons did not segregate into a row-specific pattern nor target the dPrV with a topographic pattern. Following periphery-driven molecular repatterning to a maxillary-like identity, mandibular neurons partially redirected their central projections from dPrV to vPrV. Thus, while able to induce maxillary-like molecular features resulting in vPrV final targeting, a spatially ordered lower jaw ectopic whisker pattern is insufficient to impose row-specific pre-target organisation of the central mandibular tract or a whisker-related matching pattern of afferents in dPrV. These results provide novel insights into periphery-dependent versus periphery-independent mechanisms of trigeminal ganglion and brainstem patterning in matching whisker topography.

Nordihydroguaiaretic Acid Extends the Lifespan of Drosophila and Mice, Increases Mortality-Related Tumors and Hemorrhagic Diathesis, and Alters Energy Homeostasis in Mice.

Mesonordihydroguaiaretic acid (NDGA) extends murine lifespan. The studies reported here describe its dose dependence, effects on body weight, toxicity-related clinical chemistries, and mortality-related pathologies. In flies, we characterized its effects on lifespan, food consumption, body weight, and locomotion. B6C3F1 mice were fed AIN-93M diet supplemented with 1.5, 2.5, 3.5, or 4.5 g NDGA/kg diet (1.59, 2.65, 3.71 and 4.77 mg/kg body weight/day) beginning at 12 months of age. Only the 3.5 mg/kg diet produced a highly significant increase in lifespan, as judged by either the Mantel-Cox log-rank test (p = .008) or the Gehan-Breslow-Wilcoxon test (p = .009). NDGA did not alter food intake, but dose-responsively reduced weight, suggesting it decreased the absorption or increased the utilization of calories. NDGA significantly increased the incidence of liver, lung, and thymus tumors, and peritoneal hemorrhagic diathesis found at necropsy. However, clinical chemistries found little evidence for overt toxicity. While NDGA was not overtly toxic at its therapeutic dosage, its association with severe end of life pathologies does not support the idea that NDGA consumption will increase human lifespan or health-span. The less toxic derivatives of NDGA which are under development should be explored as anti-aging therapeutics.

Somatostatin and corticotrophin releasing hormone cell types are a major source of descending input from the forebrain to the parabrachial nucleus in mice.

The pontine parabrachial nucleus (PBN) receives substantial descending input from higher order forebrain regions that exerts inhibitory and excitatory influences on taste-evoked responses. Somatostatin (Sst) and corticotrophin releasing hormone (Crh) reporter mice were used in conjunction with injection of the retrograde tracer CTb-488 into the caudal PBN to determine the extent to which Sst and Crh cell types contribute to the descending pathways originating in the lateral hypothalamus (LH), central nucleus of the amygdala (CeA), bed nucleus of the stria terminalis (BNST), and insular cortex (IC). Five to 7 days following injections, the animals were euthanized and tissue sections prepared for confocal microscopy. Crh cell types in each forebrain site except IC project to the PBN with the greatest percentage originating in the BNST. For Sst cell types, the largest percentage of double-labeled cells was found in the CeA followed by the BNST. Few retrogradely labeled cells in the LH coexpressed Sst, whereas no double-labeled cells were observed in IC. The present results suggest that Sst and Crh cell types are a substantial component of the descending pathways from the amygdala and/or BNST to the PBN and are positioned to exert neuromodulatory effects on central taste processing.

Mouse models of human cancer.

The Helmholtz Alliance Preclinical Comprehensive Cancer Center (PCCC; www.helmholtz-pccc.de) hosted the "1st International Kloster Seeon Meeting on Mouse Models of Human Cancer" in the Seeon monastery (Germany) from March 8 to 11, 2014. The meeting focused on the development and application of novel mouse models in tumor research and high-throughput technologies to overcome one of the most critical bottlenecks in translational bench-to-bedside tumor biology research. Moreover, the participants discussed basic molecular mechanisms underlying tumor initiation, progression, metastasis, and therapy resistance, which are the prerequisite for the development of novel treatment strategies and clinical applications in cancer therapy.

Effects of coumestrol administration to maternal mice during pregnancy and lactation on immunoblogulin A-secreting cells in mammary glands.

Mortality and morbidity of neonates continue to be major problems in humans and animals, and immunoblogulin A (IgA) provides protection against microbial antigens at mucosal surfaces. The present study was conducted to clarify the effects of coumestrol administration to maternal mice during pregnancy and lactation on IgA antibody-secreting cells (ASC) in mammary glands in lactating mice. From 6.5 to 16.5 days post coitus and 1 to 13 days post partum (dpp), maternal mice were administered coumestrol at 200 µg/kg body weight/day. Coumestrol administration increased the number of IgA ASC and the messenger RNA expression of IgA C-region and vascular cell adhesion molecule-1 in mammary glands of maternal mice at 14 dpp, but coumestrol administration had no effect on the number of IgA ASC in the ileum. Coumestrol administration increased serum IgA concentration in maternal mice at 14 dpp, but IgA concentrations in serum, stomach contents, intestine and feces of neonatal mice were not affected by treatment. These results imply that coumestrol administration to maternal mice during pregnancy and lactation is effective in increasing the numbers of IgA ASC in mammary glands during lactation owing to the activated messenger RNA expressions of IgA C-region and vascular cell adhesion molecule-1 in mammary gland.

[Difference of hypaconitine concentration in serum between cold-deficiency and normal mice].

OBJECTIVE: To investigate the difference of hypaconitine concentration in serum between normal and cold-deficiency mice after administration of aconite decoction. To analyze how the toxic dose of aconite decoction correlate to the metabolic environment. METHOD: Prepared cold-deficiency mice model, treated normal and cold-deficiency mice with aconite decoction for 14 days continuously, and then detected hypaconitine concentration in serum by HPLC along with survival ratio of mice on the first, seventh and fourteenth day. RESULT: After administration of aconite decoction for 14 days, the hypaconitine concentration in serum of cold-deficiency mice is close to that in normal mice. It showed aconite decoction has the ability of regulating metabolism environment, the hypaconitine concentration in serum of normal mice was higher on the seventh and fourteenth day than that on first day. It showed that aconite decoction can disturb metabolism environment of normal mice. It was also been observed that the range of variation of hypaconitine concentration in cold-deficiency mice was minor than that in normal mice during the fourteen days' administration. CONCLUSION: The difference of serum concentration in normal and cold-deficiency mice showed that there were different metabolic environments in two mice models, and the metabolic environment changed during administration. These results showed that the different toxic doses of aconite decoction were partially due to the different metabolic environments.

Sensitivity of the mouse to changes in azimuthal sound location: angular separation, spectral composition, and sound level.

Auditory spatial acuity was measured in mice using prepulse inhibition (PPI) of the acoustic startle reflex as the indicator response for stimulus detection. The prepulse was a "speaker swap" (SSwap), shifting a noise between two speakers located along the azimuth. Their angular separation, and the spectral composition and sound level of the noise were varied, as was the interstimulus interval (ISI) between SSwap and acoustic startle reflex elicitation. In Experiment 1 a 180 degrees SSwap of wide band noise (WBN) was compared with WBN Onset and Offset. SSwap and WBN Onset had near equal effects, but less than Offset. In Experiment 2 WBN SSwap was measured with speaker separations of 15, 22.5, 45, and 90 degrees . Asymptotic level and the growth rate of PPI increased with increased separation from 15 to 90 degrees , but even the 15 degrees SSwap provided significant PPI for the mean performance of the group. SSwap in Experiment 3 used octave band noise (2-4, 4-8, 8-16, or 16-32 kHz) and separations of 7.5 to 180 degrees . SSwap was most effective for the highest frequencies, with no significant PPI for SSwap below 8-16 kHz, or for separations of 7.5 degrees . In Experiment 4 SSwap had WBN sound levels from 40 to 78 dB SPL, and separations of 22.5, 45, 90, and 180 degrees : PPI increased with level, this effect varying with ISI and angular separation. These experiments extend the prior findings on sound localization in mice, and the dependence of PPI on ISI adds a reaction time-like dimension to this behavioral analysis.